CN102453747A - Tracing analysis method in cell of deoxyribonucleic acid (DNA) - Google Patents

Tracing analysis method in cell of deoxyribonucleic acid (DNA) Download PDF

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CN102453747A
CN102453747A CN2010105087817A CN201010508781A CN102453747A CN 102453747 A CN102453747 A CN 102453747A CN 2010105087817 A CN2010105087817 A CN 2010105087817A CN 201010508781 A CN201010508781 A CN 201010508781A CN 102453747 A CN102453747 A CN 102453747A
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cell
foreign gene
dyestuff
hochest
plasmid
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张晓玲
童海骏
戴尅戎
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Shanghai Institutes for Biological Sciences SIBS of CAS
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Shanghai Institutes for Biological Sciences SIBS of CAS
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Abstract

The invention relates to a tracing analysis method in a cell of deoxyribonucleic acid (DNA). In the invention, the tracing method in the cell of the DNA is optimized, and various color developing labels are selected to mark the DNA, nucleuses and lysosomes, so that existence and motion conditions of exogenous genes in the cell are researched by a co-localization method.

Description

Tracer analysis method in the cell of a kind of DNA
Technical field
The invention belongs to biological technical field; More specifically, the present invention relates to the interior tracer analysis method of cell of a kind of DNA.
Background technology
In biological technical field, transgenic technology is the most commonly used and requisite means.But after the transfection of carrying out foreign gene, how to observe the efficient that foreign gene gets into cell, and foreign gene also is that this area need be paid close attention in intracellular existence or motion conditions.Although can identify through pcr amplification and electrophoresis evaluation whether foreign gene changes over to; But it changes the back over to and but has no idea to learn in the intracellular situation that exists, and also promptly can't under the situation of live body, dynamically observe foreign gene as required in different steps residing position and motion conditions in cell
Carrying out cell marking through isotropic substance, resorcinolphthalein or dyestuff is also used by those skilled in the art.Yet the dyestuff that is used to carry out mark at present has many kinds, and some dyestuff has certain toxicity for cell.Have the colour contamination phenomenon between some dyestuff, usually resolving power is not high when using simultaneously more than a kind of dyestuff, and observing effect is unsatisfactory.
At present, be the focus in molecular therapy field for the research and development of non-viral gene delivery system, the bottleneck that the gene delivery carrier of effective and safe will be broken through gene therapy brings glad tidings to extensive patients.In the middle of the process of the novel non-virus carrier of research and development, confirm that non-virus carrier is a very important challenge in the intracellular rate-limiting step of different sorts.
Foreign gene is final express before, have three steps the most key: 1. cell endocytic; 2. endolysosome is escaped; 3. DNA goes into nuclear.
Distinguish marker DNA, lysosome, nucleus simultaneously but still have at present, utilize the technology of localized method research foreign gene that be total in intracellular motion.
Summary of the invention
The object of the present invention is to provide the interior tracer analysis method of cell of a kind of DNA.
In first aspect of the present invention, a kind of interior tracing method of cell of foreign gene is provided, said method comprises:
(1) with the fluorescein-labelled foreign gene of Cy5 the carrier of foreign gene (or carry), the foreign gene that will pass through mark changes in the cell;
(2) with the nucleus of dyestuff Hochest labeled cell;
(3) with the lysosome of dyestuff LysoTracker Red DND-99 labeled cell; With
(4) be reference with the nucleus of the blue markings in the cell and the lysosome of red-label, the foreign gene of observing the green fluorescence mark is in intracellular distribution or motion conditions.
In a preference, described foreign gene is present in the plasmid.
In another preference, step (2) in step (3) before; Or step (3) in step (2) before.
In another preference, described method with the fluorescein-labelled foreign gene of Cy5 is:
With the Cy5 resorcinolphthalein (preferably is Label IT Tracker TMReagent Intracellular NucleicAcid Localization Kit-Cy TM5) with after foreign gene hatches altogether, add NaCl and absolute ethyl alcohol, leave standstill, the centrifuging and taking deposition, washing back collecting precipitation obtains the fluorescein-labeled foreign gene of Cy5.
In another preference, described nuclear method with dyestuff Hochest labeled cell is:
In cell, add dyestuff Hochest (preferably being Hoechst 33342) label solution (preferably 2.0 ± 0.5 μ g/ml); Hatch altogether and (preferably hatched 2-10 minute; More preferably hatched 4-5 minute); Collecting cell and washing (preferably cleaning one time) with HBSS, the cell of acquisition nucleus labeling dye Hochest.
In another preference, described lysosomal method with dyestuff LysoTracker Red DND-99 labeled cell is:
In cell, add LysoTracker Red DND-99 label solution (preferably 100 ± 20nM); Hatch altogether and (preferably hatched 0.5-1.5 minute; More preferably hatched 1 minute); Collecting cell and washing (preferably cleaning one time) with HBSS, the cell of acquisition lysosome labeling dye LysoTracker Red DND-99.
In another preference, the foreign gene that will pass through mark changes the transfection reagent that adopts in the cell over to and is: PEI 600-CyD, PEI-25kDa or Lipofectamine TM2000 Reagent.
In another preference, the foreign gene of observing the green fluorescence mark through laser confocal microscope is in intracellular distribution or motion conditions.
In another preference, the excitation wavelength of resorcinolphthalein Cy5 is 649nm, and wavelength of transmitted light is 670nm;
The excitation wavelength of dyestuff Hoechst is 350nm, and wavelength of transmitted light is 461nm; Or
The excitation wavelength of dyestuff LysoTracker Red DND-99 is 577nm, and wavelength of transmitted light is 590nm.
In second aspect of the present invention, a kind of test kit is provided, said test kit is used for foreign gene is carried out spike in the cell, wherein contains: Cy5 resorcinolphthalein, dyestuff Hochest and dyestuff LysoTracker RedDND-99.
In another preference, also contain in the described test kit and be selected from one or more following reagent: mark damping fluid, aqua sterilisa, sodium-chlor, ethanol, washing composition or transfection reagent.
In another preference, also contain working instructions in the described test kit.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown and has utilized behind the fluorescein-labelled method spike pEGFP transfection 4h in the intracellular distribution of BMSC.
Embodiment
The inventor is through deep research; Optimized the method for spike in a kind of DNA cell; Chosen multiple colour developing mark and come marker DNA, nucleus and lysosome, be total to localized method research foreign gene in intracellular existence and motion conditions thereby utilize.
The inventor has utilized resorcinolphthalein Cy5 marker plasmid, the lysosome in the nucleus in the dyestuff Hochest labeled cell, dyestuff LysoTracker Red DND-99dye labeled cell.Can under the situation of live body, dynamically observe foreign gene as required in different steps residing position and motion conditions in cell through the dye marker system.Can comparatively comprehensively analyze the rate-limiting step of foreign gene in transfection process by this system, and reliable basis is provided improving carrier characteristics.This labeling technique is simple, reliable and stable.
Therefore, the invention provides tracing method in a kind of cell of foreign gene, said method comprises: (1) is with the fluorescein-labelled foreign gene of Cy5 or carry the carrier of foreign gene, and the foreign gene that will pass through mark changes in the cell; (2) with the nucleus of dyestuff Hochest labeled cell; (3) with the lysosome of dyestuff LysoTracker RedDND-99 labeled cell; (4) be reference with the nucleus of the blue markings in the cell and the lysosome of red-label, the foreign gene of observing the green fluorescence mark is in intracellular distribution or motion conditions.
The reagent that the present invention selects for use all can carry out the observation of microscopically by pair cell under the live body situation, pair cell has no damage.If cooperate the continuous fluorescence microscope photographic system, can observe in the whole DNA transfectional cell process, how foreign gene gets into cytolemma and nuclear process, and this has crucial meaning to researching and analysing foreign gene in intracellular rate-limiting step.
Because need be with three kinds of different materials of tense marker: foreign gene, lysosome and nucleus.This just needs to select suitable, complementary interferential dyestuff, the confidence level that influences test to avoid between different dyes " colour contamination " phenomenon taking place.The inventor finds three kinds of dyestuff Hochest through reasonably selecting and checking times without number, and LysoTracker Red DND-99 dye does not have " colour contamination " phenomenon between the Cy5.
Marking method for foreign gene has a variety of; I once attempted with quantum dot plasmid being carried out mark in the past; Find that this kind method is more loaded down with trivial details, and need to be used for tracer analysis after other follow-up test analyzes the situation of mark and confirm.And method of the present invention is comparatively easy to the method for the mark employing of foreign gene, need not unnecessary analysis confirmation and can directly be used for tracer analysis, and find that through test of many times system is relatively stable.For lysosome and nuclear marking method, equally also more convenient easy row.
As used herein, described " foreign gene " is also referred to as " foreign DNA ", and it also broadly comprises the plasmid (carrier) that carries foreign gene.
As optimal way of the present invention, described method with the fluorescein-labelled foreign gene of Cy5 is: with Label IT Tracker TMReagent Intracellular Nucleic Acid Localization Kit-Cy TM5 with after foreign gene hatches altogether, adds NaCl and absolute ethyl alcohol, leaves standstill, the centrifuging and taking deposition, and washing back collecting precipitation obtains the fluorescein-labeled foreign gene of Cy5.
As optimal way of the present invention; Described nuclear method with dyestuff Hochest labeled cell is: in cell, add Hoechst 33342 label solution (preferably 2.0 ± 0.5 μ g/ml); Hatch altogether and (preferably hatched 2-10 minute; More preferably hatched 4-5 minute), collecting cell and washing, the cell of acquisition nucleus labeling dye Hochest.
As optimal way of the present invention; Described lysosomal method with dyestuff LysoTracker Red DND-99 labeled cell is: in cell, add LysoTracker Red DND-99 label solution (preferably 100 ± 20nM); Hatch altogether and (preferably hatched 0.5-1.5 minute; More preferably hatched 1 minute), collecting cell and washing, the cell of acquisition lysosome labeling dye LysoTracker Red DND-99.
The foreign gene that will pass through mark changes cell over to and can adopt the known several different methods of those skilled in the art, when cell is prokaryotic organism such as intestinal bacteria, can adopt CaCl 2Method or locate MgCl 2Legal principle, used step is well-known in this area; If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome method etc.
As optimal way of the present invention, the foreign gene that will pass through mark changes the transfection reagent that adopts in the cell over to and is: PEI 600-CyD, PEI-25kDa or Lipofectamine TM2000 Reagent.
Any instrument and method of observing fluorescent mark and/or dye marker all can be applicable to the present invention, exists and motion conditions intracellular to observe foreign gene.As optimal way of the present invention, the foreign gene of observing the green fluorescence mark through laser confocal microscope is in intracellular distribution or motion conditions.
As optimal way of the present invention, the excitation wavelength of described resorcinolphthalein Cy5 is 649nm, and wavelength of transmitted light is 670nm; The excitation wavelength of dyestuff Hoechst 33342 is 350nm, and wavelength of transmitted light is 461nm; The excitation wavelength of dyestuff LysoTracker Red DND-99 is 577nm, and wavelength of transmitted light is 590nm.
The present invention also provides a kind of test kit that is used for foreign gene is carried out spike in the cell, contains in the said test kit: Cy5 resorcinolphthalein, dyestuff Hochest and dyestuff LysoTracker Red DND-99.
Any other all can be included in the described test kit for dyeing or transfection useful reagent, and other cultivation useful reagent or substratum for the duplicating of plasmid, cell also can be included in the described test kit.For example, also contain in the described test kit and be selected from one or more following reagent: mark damping fluid, aqua sterilisa, sodium-chlor, ethanol, washing composition or transfection reagent.
As optimal way of the present invention, also contain working instructions in the described test kit.
As preferred implementation of the present invention, method of the present invention can be applicable to the analysis of gene conveying situation in the non-viral gene delivery system.Described non-viral gene delivery system for example is PEI25-kDa or PEI 600The system of-CyD transfection.Method of the present invention is utilized different resorcinolphthaleins or dyestuff marker plasmid, lysosome and nucleus respectively, thereby is easy to common location foreign gene residing position in cell, analyzes the rate-limiting step of foreign gene transfection, so as to improving non-virus carrier.This tracer analysis system operation is easy, and stable good reproducibility can be used for the tracer analysis of DNA administration.
Utilize tracer analysis method in the DNA drug cell of the present invention, the inventor has analyzed three kinds of carrier Lipo2000, PEI25-kDa and PEI 600The DNA transfection efficiency of-CyD, simultaneously with naked DNA as contrast.The inventor utilizes Cy5 marker plasmid (for green fluorescence), Hochest labeled cell nuclear (being blue-fluorescence), LysoTracker Red DND-99 dye mark lysosome (being red fluorescence).The result finds that can under the situation of live body, dynamically observe foreign gene as required in different steps residing position and motion conditions in cell through method of the present invention, observation effect is good.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press; 2002) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
In following examples, nanometer non-viral gene delivery system and carrier are compound to be transfected into the method for cell with carrier through adopting, and the carrier of observation mark is in intracellular spike situation, to analyze the effect of technical scheme of the present invention.
The conversion of embodiment 1, plasmid pEGFP, amplification and purification
1, required main agents and plant and instrument
1. plasmid: pEGFP-N1 (be called for short pEGFP, available from Invotrogen);
2. competence bacteria: DH5 α (worker is given birth in Shanghai);
3.LB substratum: the 10g Trypsin freezes, the 5g yeast extract, and 10gNaCl, with the preparation of 1L ultrapure water, the pH value is transferred to 7.2-7.4, and is subsequent use behind the high-temperature sterilization;
4.LB nutrient agar: 15g agar adds 1 liter of LB substratum, treat behind the high-temperature sterilization agar uncooled in, add microbiotic and also in super clean bench, substratum poured in the microbial culture flat board, be cooled to that to be placed on 4 ℃ of refrigerators after the curdled appearance subsequent use;
5. microbiotic: ammonia benzyl microbiotic (Sigma);
6.QIAGEN plasmid is taken out test kit (QIAGEN, Plasmid Maxi Kits) greatly;
7. water-bath: DK-8D type electric heating constant temperature tank (going up the grand experimental installation of Nereid ltd);
8. vertical shaking table: SCS-24 (ShangHai City centrifugal Machine Institute);
9. uv-spectrophotometric appearance: Nanodrop2000 (Thermo);
10. desk-top horizontal centrifuge: 5810R (Eppendorf).
2, the conversion of plasmid pEGFP
1 μ l plasmid (3-5ng/ μ l) is added 100 μ l competence bacteria DH5 α; Mixing rests on 30 minutes on ice, and competence bacteria DH5 α is put into 42 ℃ water-bath; Heat shock is put into competence bacteria DH5 α at once after 1.5 minutes and is cooled off 3-5 minute on ice; At last competence bacteria is applied to and contains on the antibiotic agar culture plate of ammonia benzyl, be placed on shaking table spend the night (37 ℃, 12-16 hour).
3, the amplification of plasmid pEGFP
From the agar culture plate, behind the picking clone, put into and contain the antibiotic LB substratum of ammonia benzyl (ammonia benzyl microbiotic final concentration: 100 μ g/ml), put into shaking table (37 ℃, 300rpm, 12-16 hour).
4, the purification of plasmid pEGFP
Collect bacterium liquid (4 ℃, 6000g 15min), opens QIAGEN and takes out test kit greatly, add the resuspended bacterium liquid precipitate of 10mlP1 after, add 10ml P2, rock mixing 4-6 time back and forth after, room temperature (15 ℃-25 ℃) leaves standstill 5min.Add the P3 of 10ml precooling, rock mixing 4-6 time at once, hatch 20min on ice.Centrifugal (4 ℃, 20000g is drawn onto another one extracting centrifuge tube with supernatant after 30min), and it is centrifugal again that (4 ℃, 20000g 30min) gets supernatant.Utilize 10mlQBT balance QIAGEN-tip500 plasmid adsorption column, then supernatant is joined adsorption column and filter.After the filtration, discard filtrating, with 30mlQC adsorption column is crossed post again and clean 2 times.Discard filterable scavenging solution QC, add 15mlQF plasmid is carried out wash-out, collect elutriant after, add the 10.5ml Virahol, mixing centrifugal (4 ℃, 15000g, 30min).Careful supernatant discarded is collected the plasmid post precipitation, uses the resuspended plasmid deposition of 5ml 70% (v/v) ethanol again, room temperature centrifugal (15000g, 10min).Obtain the plasmid post precipitation, the air-dry 5-10min of room temperature with TE damping fluid dissolving plasmid deposition, utilizes the uv-spectrophotometric appearance to measure concentration and purity again.Purity: A260/A280 should be controlled at 1.6-1.8.Being placed on-20 ℃ of refrigerators preserves.
The mark of embodiment 2, DNA
One, DNA is fluorescein-labelled
1, this tests required main agents and plant and instrument
Label?IT?Tracker TM?Reagent?Intracellular?Nucleic?Acid?LocalizationKit-Cy TM5(Mirus)；
NaCl (Shanghai City chemical reagents corporation);
Absolute ethyl alcohol (Shanghai City chemical reagents corporation);
Water-bath: DK-8D type electric heating constant temperature tank (going up the grand experimental installation of Nereid ltd);
Desk-top horizontal centrifuge: 5810R (Eppendorf);
Super clean bench: VCM-420 (BIO-HAZARD).
2, DNA is fluorescein-labelled
In the pEGFP-N1 of 10 μ l, 1 μ g/ μ l, add 10 * Labeling Buffer A (Mirus), 10 μ l, add the ultrapure water 75 μ l of sterilization again, add Label IT Tracker at last TMReagent (Mirus) 5 μ l form 100 μ l reaction systems, and in 37 ℃ of water-baths, lucifuge is hatched and reacted in 1 hour.The NaCl and the absolute ethyl alcohol of two volumes that add the 5M of 1/10 volume were placed in-20 ℃ of refrigerators after 30 minutes, and 12000rpm carries out removing in centrifugal 10 minutes the Label IT Tracker that participates in reaction TMReagent, with the ethanol washing and precipitating of 500 μ l 70% (v/v), 12000rpm collecting precipitation again after centrifugal 10 minutes, supernatant discarded.In super clean bench, dry up post precipitation, use the ultrapure water dissolution precipitation, be placed on-20 ℃ of refrigerators, keep in Dark Place.
Two, plasmid is quantum dot-labeled
1, this tests required main agents and plant and instrument
Psoralen-PEO 3-Biotin(PIERCE)；
Potassium ethanoate (Shanghai City chemical reagents corporation);
Absolute ethyl alcohol (Shanghai City chemical reagents corporation);
6% (w/v) bovine serum albumin (BSA): 300mgBSA is dissolved among the 5mlPBS;
DMSO (Shanghai City chemical reagents corporation);
Quantum dot Qdot Streptavidin Conjugates-605 (Invitrogen).
2, plasmid pEGFP's is quantum dot-labeled
Plasmid pEGFP is diluted to 0.1 μ g/ μ l with ultrapure water, under the lucifuge condition, gets plasmid solution 1/100 volume, 20mM Psoralen-PEO 3-Biotin carries out mixing, and is reaction 30 minutes under the ultraviolet lamp tube of 365nm at exciting light, and this process needs carry out on ice.Reaction uses final concentration to be the liquor kalii acetici of 0.2M and the absolute ethyl alcohol of 2 times of volumes after accomplishing, and 12000rpm was settled out the plasmid that has reacted in centrifugal 10 minutes, and (plasmid and the Psoralen-PEO that has neither part nor lot in reaction contained in the inside to supernatant discarded simultaneously 3-Biotin).With 70% ethanol deposition is cleaned again, drying precipitated back dilute with water, and utilize the uv-spectrophotometric appearance to measure its concentration, subsequent use.
The quantum dot of 1 μ M is diluted with 6% (w/v) BSA, be diluted to 20nM.Simultaneously with Psoralen-PEO 3The plasmid that-Biotin connects is diluted to 5nM to be mixed with the quantum dot equal-volume, after at room temperature lucifuge is hatched 4 hours, uses final concentration to be the liquor kalii acetici of 0.2M and the absolute ethyl alcohol of 2 times of volumes; 12000rpm was settled out the plasmid that has reacted in centrifugal 10 minutes; Supernatant discarded is simultaneously cleaned drying precipitated dilute with water afterwards with 70% ethanol again to deposition; Utilize the uv-spectrophotometric appearance to measure its concentration, be used for detecting.
Embodiment 3, plasmid and transfection reagent compound
1, transfection reagent
Transfection reagent adopts nanometer non-viral gene delivery system, and one of concrete material that uses is PEI 600-CyD (PEI 600-β-CyD), teach available from the materialogy co-worker Tang Guping of Zhejiang University.Its structural formula is (m wherein as follows 1Be 10, m 2Value be 5), and m 1: m 2=2: 1, n=16).
Figure BSA00000305447300091
Another of the concrete material that uses is PEI-25kDa, for a kind of commercial transfection reagent, available from sigma company.
Another of the concrete material that uses is Lipofectamine TM2000 Reagent are called for short Lipo2000, available from Invitrogen.
2, cells transfected
The extraction of external rat BMSC and cultivation: healthy SD rat, 250 ± 10g, 8 ± 1 weeks, after the cervical vertebra dislocation method is put to death, the alcohol disinfection in 10 minutes of putting into 75% (v/v).Rat is dissected; Take out the femur and the shin bone of rat both sides hind leg respectively, will reject attached to the muscle on the bone with surgical scissors and tweezers, with femur and shin bone cuts off then; Draw the DMEM that 3ml contains 10% (v/v) foetal calf serum with disposable 5ml syringe; Again substratum is squeezed in the medullary space of femur and shin bone, marrow is gone out, flow in the petridish along liquid.The corresponding ware cell of mouse.With the volume polishing of substratum in the petridish to 10ml.Then at 5% (v/v) CO 2In the incubator, under the saturated humidity, cultivated 4 days for 37 ℃, utilize PBS that not adherent cell is all discarded earlier when changing nutrient solution, continue to cultivate and per 3 days replacing one subcultures (10% (v/v) FBS+DMEM).During to 7 days, when cell covers with individual layer basically, with 0.25% (w/v) pancreatin 37 ℃ of digestion 1 minute down, treat that most of cellular contraction becomes circle after, add substratum and stop digestion, 1000rpm is after centrifugal 4 minutes, collecting cell.The adding substratum is blown and beaten gently cell is fully suspended, and promptly obtains former generation BMSC suspension, gets 10 μ l and carries out cell counting, again with 1 * 10 4/ cm 2The density cultivation of going down to posterity.All other experiment is all taken from the cell in 3-6 generation as primary cell.
3, PEI 600-CyD and PEI-25kDa are with compound, the transfection of pEGFP
The preparation of transfection liquid: earlier PEI-25kDa is prepared into the storage liquid of 1 μ g/ μ l with ultrapure water, with PEI 600-CyD is prepared into the storage liquid of 2.7 μ g/ μ l with ultrapure water, and it is subsequent use that plasmid is diluted to 0.05 μ g/ μ l.Then according to 4.5 μ g/ μ l PEI-25kDa=100nmol/ μ l effective " N ", 13.5 μ g/ μ l PEI 600-CyD=100nmol/ μ l is " N " effectively, and 1 μ g/ μ l plasmid=3nmol/ μ l effectively " P " calculates.Then with 40 μ l, the pEGFP of 0.05 μ g/ μ l is with PEI-25kDa or the PEI of 40 μ l of corresponding concentration (concentration of calculating according to N/P) 600-CyD carries out equal-volume to be mixed, and slowly at full throttle shakes 20 seconds on dropping and the earthquake device, leaves standstill under the room temperature 30 minutes, adds 1ml serum free medium DMEM again, and mixing leaves standstill after 5 minutes available.For BMSC cell, PEI-25kDa:N/P=10; PEI 600-CyD:N/P=30.
With the BMSC cell with 1 * 10 5/ hole, every hole 1ml kind is in 12 orifice plates.After the kind plate 24 hours, cell density discards substratum near more than 90%, cleans one time with PBS and adds transfection liquid (every hole 1ml) afterwards.
The method of use Lipo2000 can be referring to the working instructions of Invitrogen company.
Three, in the mixture behind the plasmid DNA transfection in intracellular tracer analysis
1, this tests required main agents and plant and instrument
HBSS damping fluid (Invitrogen);
Lipo2000(Invitrogen)；
Hoechst?33342?labeling?solution(Invitrogen)；
LysoTracker?Red?DND-99?labeling?solution(Invitrogen)；
Cell culture incubator: HerA cell 150 (Heraeus);
Super clean bench: VCM-420 (BIO-HAZARD);
Laser confocal microscope: Tcs sp5 (Leica).
2, in the mixture behind the plasmid DNA transfection in intracellular spike
PEI 600-CyD organizes N/P=30, and PEI-25kDa organizes N/P=10.After the transfection 4 hours, supernatant discarded adds 2.0 μ g/ml Hoechst, 33342 label solution, hatches 5 minutes for 37 ℃.Supernatant discarded, and carry out twice cleaning with HBSS damping fluid pair cell adds 100nM LysoTracker Red DND-99 label solution again, at room temperature hatch 1 minute after, supernatant discarded, clean twice with HBSS after, carry out the observation of laser confocal microscope.The excitation wavelength of quantum dot is 488nm, and wavelength of transmitted light is 605nm.The excitation wavelength of resorcinolphthalein Cy5 is 649nm, and wavelength of transmitted light is 670nm.The excitation wavelength of Hoechst 33342 is 350nm, and wavelength of transmitted light is 461nm.The excitation wavelength of LysoTracker Red DND-99 is 577nm, and wavelength of transmitted light is 590nm.
See Fig. 1 in intracellular tracer analysis result behind the plasmid DNA transfection in the mixture.Green is the plasmid of the fluorescein-labelled mark of Cy5 among the figure, and blueness is the painted nucleus of Hochest, and redness is the painted lysosome of LysoTrackerRed DND-99 dye.
The row of going up of Fig. 1 is that the fluorescein-labeled plasmid of taking under 20 times of object lens also has the painted lysosomal composite effect figure of LysoTracker Red DND-99 dye with the painted nucleus of Hochest, and following row then is the photo of taking under 40 times of object lens.Therefrom can find in the Control group, all do not have plasmid in tenuigenin and the nucleus, explain that the gymnoplasm grain of Control can't get into cell, more can't get into nucleus.And in the Lipo2000 group, find to have some plasmids and get into cell, but quantity seldom, almost do not have and get into nuclear plasmid, reviews PEI25-kDa group (N/P=10) and PEI 600-CyD organizes (N/P=30), finds that the plasmid that in tenuigenin and in the nucleus, occurs all will increase significantly.Though in these two groups, state of aggregation has appearred in the plasmid of some, these plasmids all are in the extracellular, be likely to have been remained in the extracellular matrix, and cohesion has taken place.
Discuss and analyze
After carrying out the foreign gene plasmid of foreign gene (or have) transfection, can analyze DNA very intuitively in intracellular distribution through above spike system.For example in the application's embodiment; When the efficient of the entering cell that is used to identify transfection reagent and empty plasmid, can see that for gymnoplasm grain group, the DNA ratio is easier to condense; Be difficult to pass through cytolemma and get into tenuigenin; And the non-virus carrier of this liposome type of utilization Lipo2000 finds that the situation of plasmid generation cohesion is slowed down to some extent, and the part DNA can pass through the cytolemma of cell; But behind the entering lysosome, fail to take place the lysosome escape and can't get into nucleus performance biological effect.And for PEI-25kDa and PEI 600These two types of non-virus carriers of-CyD, the plasmid in the mixture can pass through cytolemma more effectively and get into tenuigenin, and in nucleus, also can find the existence of a lot of plasmids, proves PEI-25kDa and PEI 600-CyD can bring into play certain " proton sponge effect ", and part DNA generation endolysosome is escaped, thereby gets into nucleus performance biological effect.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (10)

1. tracing method in the cell of a foreign gene is characterized in that said method comprises:
(1) with the fluorescein-labelled foreign gene of Cy5, the foreign gene that will pass through mark changes in the cell;
(2) with the nucleus of dyestuff Hochest labeled cell;
(3) with the lysosome of dyestuff LysoTracker Red DND-99 labeled cell; With
(4) be reference with the nucleus of the blue markings in the cell and the lysosome of red-label, the foreign gene of observing the green fluorescence mark is in intracellular distribution or motion conditions.
2. the method for claim 1 is characterized in that, described foreign gene is present in the plasmid.
3. the method for claim 1 is characterized in that, described method with the fluorescein-labelled foreign gene of Cy5 is:
After Cy5 resorcinolphthalein and foreign gene hatched altogether, add NaCl and absolute ethyl alcohol, leave standstill, the centrifuging and taking deposition, washing back collecting precipitation obtains the fluorescein-labeled foreign gene of Cy5.
4. the method for claim 1 is characterized in that, described nuclear method with dyestuff Hochest labeled cell is:
In cell, add dyestuff Hochest label solution, hatch altogether, collecting cell and washing, the cell of acquisition nucleus labeling dye Hochest.
5. the method for claim 1 is characterized in that, described lysosomal method with dyestuff LysoTracker RedDND-99 labeled cell is:
In cell, add LysoTracker Red DND-99 label solution, hatch altogether, collecting cell and washing, the cell of acquisition lysosome labeling dye LysoTracker Red DND-99.
6. the method for claim 1 is characterized in that, the foreign gene that will pass through mark changes the transfection reagent that adopts in the cell over to and is: PEI 600-CyD, PEI-25kDa or Lipofectamine TM2000Reagent.
7. the method for claim 1 is characterized in that, the foreign gene of observing the green fluorescence mark through laser confocal microscope is in intracellular distribution or motion conditions.
8. method as claimed in claim 7 is characterized in that,
The excitation wavelength of resorcinolphthalein Cy5 is 649nm, and wavelength of transmitted light is 670nm;
The excitation wavelength of dyestuff Hoechst is 350nm, and wavelength of transmitted light is 461nm; Or
The excitation wavelength of dyestuff LysoTracker Red DND-99 is 577nm, and wavelength of transmitted light is 590nm.
9. a test kit is characterized in that, said test kit is used for foreign gene is carried out spike in the cell, wherein contains: Cy5 resorcinolphthalein, dyestuff Hochest and dyestuff LysoTracker Red DND-99.
10. test kit as claimed in claim 9 is characterized in that, wherein also contains to be selected from one or more following reagent:
Mark damping fluid, aqua sterilisa, sodium-chlor, ethanol, washing composition or transfection reagent.
CN2010105087817A 2010-10-18 2010-10-18 Tracing analysis method in cell of deoxyribonucleic acid (DNA) Pending CN102453747A (en)

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Application publication date: 20120516